Human development begins when an egg is fertilized by a sperm. This event activates the first round of genome duplication, after which the resulting 2-cell embryo activates the expression of genes in the newly formed zygote. Five more rounds of cell division and a blastocyst appears, marking the beginning of cell differentiation: trophoblasts stem (TS) cells on the spherical surface of the blastocyst will give rise to the placental, and embryonic stem (ES) cells inside the blastocyst will give rise to the embryo. During this process, a remarkable and very rare event occurs in which TS cells differentiate into trophoblast giant (TG) cells that are required for implantation of the embryo into the uterine lining. During all of the trillions of cell divisions required to make an adult human, genome duplication is restricted to once per cell division, but in TG cells and in megakaryocytes, endoduplication occurs, a process whereby the genome is duplicated multiple times without undergoing cell division. We are taking advantage of our background in DNA replication to focus on three questions concerning preimplantation mammalian development: How is DNA replication activated in fertilized eggs? What genes are involved in the production and subsequent differentiation of TS and ES cells? What triggers endoreduplication during differentiation of TS to TG cells?[unreadable] [unreadable] During the past year, we have made the following discoveries:[unreadable] [unreadable] 1) Dickkopf-like 1 (DkkL1) is a gene that is related to the Dickkopf gene family which is a group of proteins that are characterized as secreted antagonists of Wingless signal transduction proteins. DkkL1 mRNA is found in preimplantation mouse embryos and in developing neural tissue, but in adults it is found primarily in the testes. In an effort to elucidate its function, the distribution of Dickkopf-like 1 protein (DkkL1) in mouse testis and mature sperm was analyzed by immuno-histochemistry and immuno-blotting techniques. DkkL1 first appeared in the developing spermatocytes in seminiferous tubules as early as Stage XII, coincident with the appearance of DkkL1 mRNA. Surprisingly, however, DkkL1 localized to the developing acrosome in spermatocytes and spermatids and to the acrosome in mature sperm. Furthermore, DkkL1 was N-glycosylated in the testis, but it did not appear to be excreted, and the DkkL1 in mature sperm was no longer N-glycosylated, suggesting that additional post-translational modifications occurred during the final stages of spermatogenesis. These results identify a member of the Dickkopf family as a novel acrosomal protein that may be involved in acrosome assembly or function, a unique role for a secreted signaling molecule.[unreadable] [unreadable] 2) The La protein is a target of autoantibodies in patients suffering from Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La+/-intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM.[unreadable] [unreadable] For additional information, visit our web site at (http://depamphilislab.nichd.nih.gov/), and read our three recent publications.[unreadable] [unreadable] Kohn, M.J., K.J. Kaneko and M.L. DePamphilis (2005) DkkL1(Soggy), A Dickkopf Family Member, Localizes To The Acrosome During Mammalian Spermatogenesis, Mol. Reprod. Dev. 71:516-522.[unreadable] [unreadable] Park, J-M., M. J. Kohn, M. Bruinsma, C. Vech, R.V. Intine, S. Fuhrmann, A. Grinberg, I. Mukherjee, P.E. Love, M.S. Ko, M.L. DePamphilis and R.J. Maraia. (2006) The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells., Mol. Cell. Biol. 26:1445-51.[unreadable] [unreadable] Kohn, M.J., Kaneko, K.J., Yagi, R., Litscher, E.S., Wassarman, P.M., DePamphilis, M.L. (2006) Dickkopf-like 1- a Protein Unique to Mammals that is Associated Both With Formation of Trophoblast Stem Cells and With Spermatogenesis. In ?Y Chromosome and Male Germ Cell Biology? (Lau, Y.-F.C. and Chan, W.Y., eds), World Scientific Publishers, Hackensack, NJ, in press.